The production of exogenous proteins using gene recombination technology is used in various industries. The hosts used for their production consist mainly of E. coli, yeast, animal cells, plant cells and insect cells. However, a host has yet to be developed that is capable of efficiently producing all kinds of exogenous proteins and, as it is necessary to construct a production system for each target protein, a technical breakthrough is being sought in technology for producing exogenous proteins in individual hosts.
Systems using bacteria like E. coli or yeast have problems with posttranslational modification, and in some cases these systems are unable to synthesize proteins in a form that allows them to function adequately. In addition, although animal cells allow proteins to be synthesized in a functional form, it is typically difficult to grow these cells and the production volume is low thereby making this uneconomical.
On the other hand, in the production of gene recombinant proteins using insects or insect cells, useful proteins having enzymatic or physiological activity can be produced comparatively inexpensively and modifications can be obtained, following protein translation, that resemble those in animals. More specifically, a method in which a baculovirus incorporated a recombinant exogenous gene is infected into insects or insect cells allows the exogenous protein to be produced comparatively inexpensively, and physiologically active proteins are known that have been commercialized as pharmaceuticals (Japanese Unexamined Patent Publication Nos. 61-9288 and 61-9297).
In the case of the production of cytokines, which are physiologically active substances having immunoregulatory functions and which are attracting attention in pharmaceutical applications, methods are disclosed in Japanese Unexamined Patent Publication Nos. 3-139276 and 9-234085 in which silkworms are inoculated with BmNPV containing a feline interferon-ω gene and a canine interferon-γ gene, respectively. In addition, a process for producing human collagen using insect cells infected with baculovirus is known as an example of the production of a protein other than interferon using insects (Japanese Unexamined Patent Publication No. 8-23979).
However, as technologies for producing recombinant proteins using insects or insect cells of the prior art use a recombinant virus to incorporate an exogenous gene, there is the problem of the need for deactivation or containment of recombinant virus from the viewpoint of safety. In addition, in methods in which a recombinant virus is inoculated into a silkworm, as the inoculation of the recombinant virus is troublesome task and the target exogenous protein is produced in silkworm hemolymph, it is necessary to purify the target recombinant protein from the large amount of contaminating proteins originating in the body fluids of the silkworm. Consequently, there was the problem of it being difficult to obtain a highly pure recombinant protein.
On the other hand, attempts have been made in recent years to recombine exogenous genes into insect chromosomes, and a method has been developed that uses homologous recombination to introduce and express in silkworm chromosomes a fused gene in which jellyfish green fluorescence-protein gene was coupled to silkworm fibroin L chain gene using DNA of Autographa californica nuclear polyhedrosis virus (AcNPV), which is a type of nuclear polyhedrosis virus (Genes Dev., 13, 511-516, 1999), and a silkworm containing human collagen gene and a production process have been disclosed that utilize this technology (Japanese Unexamined Patent Publication No. 2001-161214). Recently, research has been conducted on a method for expressing a protein encoded by an exogenous gene by stably introducing that exogenous gene into silkworm chromosomes using piggyBac, which is a transposon originating in a lepidopteron, using the jellyfish green fluorescence protein as a model, and the gene has been confirmed to be stably propagated to offspring by mating (Nature Biotechnology, 18, 81-84, 2000).
However, in the aforementioned method for inserting an exogenous protein gene into insect chromosomes using AcNPV, as a recombinant baculovirus (AcNPV) is used, there is still the problem of having to deactivate and contain the recombinant virus. In the example that used the piggyBac transposon, as the amount of green fluorescence protein produced is inadequate and as it is also produced throughout the silkworm, sophisticated purification technology is required to recover the expressed recombinant green fluorescence protein in a highly pure form, thereby resulting in the method being uneconomical. In addition, the amount of recombinant protein produced is inadequate and extremely low.
Namely, in this technology for producing an exogenous protein using insect cells as a host, there are several problems such as the need to deactivate and contain the recombinant baculovirus, the difficulty in purifying the target protein from body fluid in which a large amount of contaminating proteins are present, as in the case of using silkworms, and the expressed amount of the target protein being low.
There are no known examples, thus far, of expressing a target protein by inserting a gene that encodes a physiologically active protein such as a cytokine gene into silkworm chromosomes. In addition, there are also no examples of having recovered a recombinant cytokine from a site, other than silkworm body fluid, such as a silk gland or silk thread secreted by silkworms, and confirming the physiological activity of the resulting cytokine. In addition, there are also no precedents regarding a silkworm capable of inheriting such properties. In addition, there are no examples of having produced a large amount of recombinant protein in silk thread using a recombinant silkworm produced using a transposon.